Synthesis Of Tea Flavor Substance Theanine Via Escherichia Coli Microorganisms

Theanine,a unique flavor substance in tea,as a very representative non-protein amino acid in tea tree,is one of the fresh flavor components of tea leaves.It has a much longer history and culture,and its taste is more natural and sweet,more in line with the contemporary pursuit of the concept of a healthy diet.However,what should not be overlooked is the unique flavour substance theanine,a highly representative non-protein amino acid of the tea tree,which is one of the fresh flavour components of tea leaves.It has also been frequently used in the food,pharmaceutical and health care,and daily chemical industries because of its outstanding health functions.Recently,there has been a lot of research on L-theanine.This study involved the production of L-theanine by whole-cell catalysis and microbial fermentation.γ-glutamine methylamine synthase from Methyloversatilis universalis FAM5 and a polyphosphate kinase from Escherichia coli H736 were coupled to construct a genetically engineered strain to catalyze the synthesis of L-theanine.As well as including heterologous expression of the genes for alanine transaminase derived from Bacillus and alanine decarboxylase derived from Camellia sinensis,for yeast glutamine permease derived from Saccharomyces cerevisiae,L-theanine was produced by microbial one-step fermentation with glucose as substrate.The main points are as follows:(1)Construction of an ATP regeneration system to enhance theanine biosynthesis.The optimum conditions for the production of E.coli FD01 were found to be 30°C,p H 7.0,0.5mmol/L for the addition of IPTG and 200 mmol/L for the addition of the substrates Lglutamic acid and ethylamine.The optimum production conditions for E.coli FD02 were:temperature 37°C,p H 7.0,optimum addition of the inducer IPTG at 0.3 mmol/L,and addition of the substrates L-glutamic acid and ethylamine at a concentration of 200 mmol/L.The results indicated that the L-theanine production efficiency of E.coli FD02 was increased by13.4% relative to that of E.coli FD01.This indicates that the construction of the ATP regeneration system has been successfully demonstrated to enhance L-theanine biosynthesis in this experiment.The whole-cell catalytic reaction of the medium-sized reaction system was also performed on the engineered bacterium E.coli FD02,with a yield of 1.86 g/L of Ltheanine.(2)Enhanced theanine synthesis by theanine transporter protein overexpression.By comparing the L-theanine production of genetically engineered strain E.coli FD03 with that of genetically engineered strain E.coli FD04,it was found that the L-theanine yield of the latter was 14.7% higher than that of the former,with a maximum progressive cumulative yield of 2.9 g/L of L-theanine.Microbial fermentation achieved a 5% sugar conversion rate,enabling the production of L-theanine by one-step fermentation of glucose.It was also confirmed that theanine transporter protein overexpression enhanced L-theanine synthesis.The maximum stepwise cumulative yield of L-theanine in the medium-sized reaction system was 2.9 g/L.The fermentation enabled the production of L-theanine by one-step glucose fermentation.(3)Bioinformatics analysis of γ-glutamylmethylamine synthase GMAS.In this study,the bioinformatic analysis of γ-glutamylmethylamine synthetase GMAS was carried out to analyse the basic physicochemical properties and structure of GMAS protein,which indicates that E.coli GMAS protein is a non-secretory protein and is a stable hydrophilic protein.It is also not a transmembrane protein.The results of the functional domain analysis indicate that the GMAS protein has a conserved structural domain and this information provides an important basis for the production of heterologous expression of the GMAS protein.In other words,it will provide a high-level technical guide for in-depth research into the industrial production of L-theanine.